Use of a porous calcium carbonate based material as support of a growth factor in the preparation of a bioabsorbable implant

ABSTRACT

A method for treating a living organism includes fitting in the living organism a bioabsorbable implant. The bioabsorbable implant includes a support of porous calcium carbonate based material for supporting at least one growth factor. The calcium carbonate based support material forms an external wall of the support.

FIELD OF THE INVENTION

The subject of the invention is the use of a calcium carbonate basedmaterial as support for a growth factor in the preparation of abioabsorbable implant intended to be fitted in a living organism.

Such a material can be used, in particular, as a bone-formation implant.

BACKGROUND

It is known that bone surgery often requires the fitting of bone graftsor implants constituting artificial replacements for such grafts.

Carrying out allografting raises objections, for obvious reasons ofpublic health, in view of the risks of transmission of certain seriousviral illnesses or illnesses caused by unconventional transmittableagents or "prions".

From this point of view, carrying out autografting is more satisfactory,but taking the graft leads to significant risks of morbidity; see, forexample, Christopher J. Damien and J. Russell Parsons, Journal ofApplied Biomaterials, Vol. 2, 187-208 (1991).

For these reasons, it has been recommended to use implants based onbiocompatible materials such as tri-calcium phosphate, hydroxyapatite,plaster, coral, polymers based on poly(lactic acid) and/or poly(glycolicacid), etc.

Some of these materials, including calcium carbonate, are bioabsorbableand allow the progressive formation of newly formed bone tissue at theexpense of the implanted material being absorbed; see especiallyEuropean Patent 0,022,724.

It is moreover known that the presence of certain osteoinductive factorsin the implants promotes bone regrowth. However, specialists have untilnow judged it to be necessary to add to the implant collagen acting as asupport for the osteoinductive factors; see in this context the articleby Damien and Russell Parsons already cited hereinabove and PatentApplication FR-2,637,502.

SUMMARY OF THE INVENTION

It has now been discovered that a porous calcium carbonate basedmaterial such as coral can act as a support for osteoinductive factors,and more generally for growth factors, in the preparation ofbioabsorbable implants and that the presence of collagen is neithernecessary nor desirable in the case when the implant is intended to beused as a bone-formation implant.

DESCRIPTION OF PREFERRED EMBODIMENTS

The subject of the present invention is therefore the use of a porouscalcium carbonate based material as support for at least one growthfactor in the preparation of a bioabsorbable implant intended to befitted in a living animal organism, in particular in a vertebrate,including humans, the said support being free of collagen in the casewhen the said growth factor is an osteoinductive factor.

When the growth factors are osteoinductive factors, the implants can beused as bone-formation implants. Of course, these implants may contain,in addition to the osteoinductive factors, other growth factors. Theimplants thus obtained can either be fitted as bone fillers which can beabsorbed progressively in favour of newly formed tissue or implanted ina non-osseous site, for example connective tissue, where they give riseto bone tissue which can subsequently be used as a bone autograftmaterial.

When the calcium carbonate support is loaded with a non-osteoinductivegrowth factor, it can be used in particular as a support for in vivoculture of living cells. Depending on the cells and the growth factorsused, the implant, after fitting, may be used in particular as a supportfor obtaining tissue newly formed on the implantation site. This newlyformed tissue can be used as a replacement for defective organ parts(pancreas/intestine connection, urethra, bladder, pericardium, etc.).

The implant of the invention can also be used as an in vivo culturesupport for cells which have been genetically modified, especially byinsertion of a suitable gene, in a manner which is known per se, inorder to remedy a genetic defect. The modified cells may also come fromautologous sampling. The implants thus obtained constitute an "organoid"serving as a therapeutic treatment device, fitting of which makes itpossible to make up for a defective organ, and in particular to remedycertain dysfunctions of genetic origin. This is therefore a novel way ofimplementing genetic therapies.

For example, a hollow coral piece may be made, provided with an openingwhich can be closed off by a screwed or press-fitted plug which is alsomade of coral. For example, for implantation in humans, this may be ahollow cylinder having a height of from 2 to 10 cm, an external diameterof from 1 to 3 cm and an internal diameter of from 0.5 to 2 cm. Theinternal wall may be impregnated with a collagen loaded with growthfactors such as TGF-beta. The external wall may be impregnated with asolution of an angiogenic factor (FGF). The modified autologous cells(for example fibroblasts) are previously cultivated in vitro on a solidsupport such as a network of collagen fibres or coral granules. Theculture of modified cells is introduced, with its solid support, intothe hollow part of the implant. The latter is then closed off using theplug and the implantation, for example intra-abdonimal, is carried out.The implanted cells become organized into a vascularized tissuesurrounded by a connective tissue replacing the coral as it is resorbed.

The cells introduced into the implant may be undifferentiated cells, thedifferentiation of which is carried out using endogenous or exogenouscytokines.

The calcium carbonate based material usable as a support for growthfactors is preferably aragonite. Use may, in particular, be made ofcoral skeleton. One of the advantages of using coral skeleton is that itcontains communicating pores which promotes neovascularization as wellas invasion of the material by bone cells or other cells previouslyintroduced or recruited in situ.

A material having pore diameters lying between 50 and 500 microns, inparticular between 200 and 400 microns, is preferably used as supportmaterial. The porosity, that is to say the proportion by volume of thepores as a ratio of the total volume of the material generally liesbetween 20 and 80%, for example between 40 and 60%.

Materials of this type can be obtained by using, in particular, coral ofthe genera Porites, Acropora, Goniopora, Lobophyllia, Simphyllia andMillipora.

It is known that growth factors are polypeptides which act locally oncertain cells and influence their proliferation, their migration and/ortheir differentiation or which stimulate the production of other growthfactors.

Numerous growth factors are currently known.

For example, growth factors having an osteoinductive effect are knownand their effects have been studied, in particular in vitro, on culturesof various bone cells.

Among growth factors whose activity is specific to the osseous medium,mention may be made of certain proteins of demineralized bone, or DBM(Demineralized Bone Matrix) and in particular the proteins called BP(Bone Protein) or BMP (Bone Morphogenetic Protein) which actuallycontain a plurality of constituents; in humans, for example, theconstituents called osteonectin and osteocalcin have, in particular,been isolated. Another active protein is osteogenin.

Among the growth factors influencing cellular growth in general,including the growth of bone cells, mention may in particular be made ofsomatomedins or IGF (Insulin-like Growth Factor), PDGF (Platelet-DerivedGrowth Factor), FGF (Fibroblast Growth Factor), BDGF II (orbeta-2-microglobulin), TGFs (Transforming Growth Factor), in particularTGF-beta (or bTGF) etc.

The usable doses of growth factors are known or can be determined byroutine experiments on animal models. Use is generally made of growthfactors whose action is local, at doses of a few micrograms or of a fewtens of micrograms.

The invention also relates to the preparation of porous calciumcarbonate supports loaded with growth factors. The latter can beincorporated

by impregnation, optionally followed by drying, using a solution or agel containing the growth factor,

by deposition of films by capillary action using a solution of thegrowth factor by binding with the calcium carbonate by means of acoating with electronegative surface potential (heparin, heparansulphate, dextrane sulphate, dermatane sulphate, xylane sulphate, etc.).

It is possible to facilitate fixing and retention of the growth factorson the calcium carbonate support by applying them in the form of acombination with a binder, especially a binder capable of forming a gel.Such binders are known. They are, for example, collagen or cellulosederivatives such as methylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, etc. Theseproducts are biocompatible. They are commercially available. Use mayalso be made, as binders, of a fibrin precursor, for example fibrinogenor a biological adhesive (cryoprecipitate).

The binder has, in particular, the effect of promoting fixing of thegrowth factor and optionally its progressive release.

The binder is generally progressively absorbed as the implant isreincorporated by a newly formed tissue.

It has, however, been observed that, in the case of a bone-formationimplant, the presence of collagen as binder does not promote thecreation of new bone tissue but would, however, tend to retard it.

The following examples illustrate the invention.

EXAMPLE 1

Coral discs having a diameter of 6 mm and a thickness of 2 mm (origin:INOTEB) is used as starting material.

A bovine BP having a protein concentration of 1.52 mg/ml is used asosteoinductive factor.

The BP is diluted so as to obtain doses of 10, 35 and 100 micrograms ofactive principle in a volume of 20 microliters. Each coral disc isimpregnated with 20 microliters of one of the solutions obtained. Afterinfiltration of the solution into the pores, the discs are frozenrapidly then subjected to freeze-drying.

With the discs thus treated, subcutaneous implants are made in theventral region in rats. Use is made of three groups of five animalswhich each receive a coral implant impregnated with 10, 35 or 100micrograms of BP, respectively.

After 27 days, a dose of 20 mg/kg of a labelling agent consisting ofcalcein is administered subcutaneously to the animals. On thetwenty-eighth day, the implants are extracted, weighed, photographed andfixed in methanol. The samples are coated in poly(methyl methacrylate)with a view to histological examination. The samples are sectioned andcoloured with the reagent Rapid Bone Stain and with Van Gieson'spicro-fuchsin. The sections are examined under a microscope.

The weights of the coral discs before implantation and afterexplantation are indicated in Table 1 below, in which the results areexpressed in the form of a mean ± standard deviation.

                  TABLE 1                                                         ______________________________________                                               Weight of the discs (mg)                                                       before       after                                                    Group   implantation explantation                                                                            weight gain                                    ______________________________________                                        I        86.0 ± 11.2                                                                             97.2 ± 13.0                                                                         11.2 ± 6.0                                  II      84.4 ± 4.1                                                                              114.2 ± 14.2                                                                         29.8 ± 12.1                                 III     83.2 ± 9.5                                                                                128 ± 12.2                                                                         45.4 ± 10.1                                 ______________________________________                                    

In group I, examination under a microscope shows a formation of bonewhich is still immature and haematopoietic marrow throughout theimplant, as well as the formation of bone at the surface. The bone formsand conduction by the material allows growth of the bone. There is abalance between osteoinduction and osteoconduction.

In group II, mineralization is good throughout the implant, the newlyformed bone is mature and covers the coral. Osteoinduction predominatesover osteoconduction and there is more bone on the periphery of theimplant.

In group III, the samples have a thick outer edge of mature bone withhaematopoietic marrow at the center. In several cases, cartilage andcartilage undergoing mineralization are observed after a few weeks.

EXAMPLE 2

Trephination is carried out on rats by removal of a circular button witha diameter of 8 mm from the top of the cranium. A coral disc having adiameter of 8 mm is applied into the window thus obtained. The coraldiscs are impregnated in a manner similar to that described in thepreceding example, by the osteoinductive factor (recombinant humanBMP-2), in a ratio of 0.5 microgram of BMP-2 to 1 mg of coral. On theday of the operation, calcein is injected intramuscularly in aproportion of 20 mg/kg. On the twenty-seventh day, xylenol orange (90mg/kg) is injected intramuscularly.

These dyes give a yellow and orange fluorescence outlining the boneformations.

On the twenty-eighth day, a part of the top of the cranium including theimplant is sampled and it is fixed in neutral 10% aqueous formaldehydesolution for histological study after inclusion in methyl methacrylateon non-demineralized sections dyed with the dye Giemsa-Paragon.Microradiographs demonstrate mineralization.

The sections show a bone formation starting from the borders of the bonelesion and extending over the edges of the coral disc.

In the case of the coral impregnated with BPM-2, bone is present overthe entire disc. Absorption of the coral is more rapid than in theabsence of BMP-2.

EXAMPLE 3

Bilateral and symmetrical trephination of the cranium is carried out onrabbits at the level of the parietal bones, by removal of two circularbuttons with a diameter of 10 mm.

Five batches of rabbits are taken: four test groups and one controlgroup. A single side is treated in the individuals of each test group.

Group 1 (symbol MF): 50 microliters of methylcellulose gel containing 1μg of TGF-beta is applied into one of the windows.

Group 2 (symbol BF): 100 μl of biological adhesive to which one 1 μg ofTGF-beta was added is applied into one of the windows.

Group 3 (symbol BCF): 50 microliters of a mixture similar to thatapplied to the BF group, but additionally containing 70 mg of Poritescoral granulates marketed under the name BIOCORAL 450 by the CompanyINOTEB is applied.

Group 4 (symbol BC): the same mixture as for group 3 was applied, butwithout growth factor.

The evolution of bone construction is followed by cranialtomodensitometry. The diameters and areas of the trephinations aremeasured on an image analyser. After sacrifice of the animals studied,after one month, the top of the cranium is sampled then prepared with amicrotome for histological analysis by histomorphometry and fluorescencemicroscopy. In order to label the bone formation surfaces, 2 ml ofoxytetraquinol were injected on the tenth and third days precedingsacrifice.

The following observations were made:

After 30 days, groups BC and BCF have completely covered the scar withan osteoid tissue.

For the other groups, covering is not complete. The diameter on thetreated side is less than the diameter on the control side. The diameteron the control side is less than the diameters observed in the controlgroup.

The bone span volume (BSV) was determined by histomorphometry.

The BCF group has the highest BSV, followed by the BC group.

Studying the animals in the groups treated with coral shows that thegranulates are absorbed progressively. Coral allowed the rapid creationof bone spans between these granulates.

In general, the addition of the growth factor increases theremineralization rate.

EXAMPLE 4

A piece of coral (porosity: 50%) is covered with a mouse collagen gel oftype I containing an angiogenic growth factor (bFGF).

By using a bFGF labelled with radioactive iodine it was possible toshow, by release tests, that the binding of the bFGF to the coralsupport is very strong. This binding is further enhanced in the presenceof heparans or heparins.

The pieces of coral thus treated are implanted subcutaneously in mice.Macroscopic and histological observation of the implants is carried outfor one year.

In a few months the coral is absorbed. It is replaced by connectivetissue of the same size and the same shape as the implant. At the end ofthree months strong vascularization and the absence of any inflammatorycell next to the coral residues is observed. The connective tissue isformed by mesenchymal cells of low density.

In other experiments, the collagen was replaced bycarboxymethylcellulose or fibrinogen.

EXAMPLE 5

A hollow coral cylinder is made.

A network of collagen fibres containing autologous fibroblastsgenetically modified by insertion of a gene using a retroviral vector isintroduced into the hollow part.

The external wall of the cylinder was previously impregnated with acollagen gel of type I associated with heparin. The coral thuspretreated is incubated in the presence of an angiogenic growth factor(bFGF). Finally, the cylinder is closed off using a coral plug andintraperitoneal implantation is carried out in a dog of mass 25 kg.

After 45 days the implant is withdrawn. The vascularization of thelatter is significant and comprises vessels of large calibre. Absorptionof the coral is initiated. The modified cells are viable and express theinserted gene.

They are organized into a vascularized tissue.

A similar experiment was carried out on mice deficient inbeta-glucuronidase and having a lysosomal accumulation disease. A humancDNA coding for beta-glucuronidase is inserted into a retroviral vectorwhich is subsequently used for modifying (autologous) mouse cells. Themodified cells are introduced into a network of collagen fibres and thewhole is introduced into a coral cylinder treated in a manner similar tothat described hereinabove. The implants thus prepared are inserted intothe mesentery of the deficient mice.

The implanted modified cells are capable of secreting the missing enzymewhich passes into the general circulation, thus leading to remission ofthe physiological consequences of the genetic defect, and in particularthe correction of the hepatic and splenic lesions.

We claim:
 1. A method of treating a living organism comprising fittingin said living organism a bioabsorbable implant having a support ofporous calcium carbonate based material supporting at least one growthfactor, said support being free of collagen in a case when said growthfactor is an osteoinductive factor and wherein said calcium carbonatebased material comprises an external wall.
 2. A method according toclaim 1, wherein said porous calcium carbonate is in the form ofaragonite.
 3. A method according to claim 1, wherein said growth factoris an osteoinductive factor.
 4. A method according to claim 3, whereinsaid bioabsorbable implant is fitted as a bone filler.
 5. A methodaccording to claim 3, wherein said bioabsorbable implant is fitted in anon-osseous site.
 6. A method according to claim 1, wherein said growthfactor is a non-osteoinductive factor.
 7. A method according to claim 6,wherein said bioabsorbable implant supports an in vivo cell culture. 8.A method according to claim 7, wherein said bioabsorbable implant is inthe form of a hollow piece provided with an opening which can be closedoff by a plug.
 9. A method according to claim 8, wherein said at leastone growth factor is deposited by impregnation on an internal wall ofsaid hollow piece.
 10. A method according to claim 8, wherein a cellculture to be cultivated is introduced into a hollow part of saidbioabsorbable implant and said hollow part is then closed off using saidplug before carrying out implantation.
 11. A method according to claim1, wherein said living organism is a vertebrate.
 12. A method accordingto claim 2, wherein said porous calcium carbonate is in the form ofcoral skeleton.
 13. A method of preparing a bioabsorbable implant havinga porous calcium carbonate support as a support for at least onenon-osteoinductive growth factor, comprising forming a porous calciumcarbonate support and applying on said calcium carbonate support asolution or a gel containing said at least one non-osteoinductive growthfactor.
 14. A method according to claim 13, wherein said solution or gelis applied by impregnation or deposition.
 15. A method according toclaim 14, wherein said deposition comprises binding said solution tosaid support by coating said support with a film having electronegativesurface potential.
 16. A method according to claim 15, wherein said filmcomprises heparin, heparan, sulphate, dextrane sulphate, dermatanesulphate or xylane sulphate.
 17. A bioabsorbable implant to be fitted ina living organism, comprising a support including a porous calciumcarbonate based material and at least one growth factor, said calciumcarbonate support containing said at least one growth factor and havingan unmodified surface.
 18. A method of treating a living organismcomprising fitting in said living organism a bioabsorbable implanthaving a support consisting essentially of a porous calcium carbonatebased material supporting at least one growth factor, said support beingfree of collagen in a case when said growth factor is an osteoinductivefactor.
 19. A bioabsorbable implant to be fitted in a living organism,comprising a support consisting essentially of a porous calciumcarbonate based material and at least one growth factor, said calciumcarbonate support containing said at least one growth factor.
 20. Themethod of claim 18, wherein said calcium carbonate is in the form ofaragonite.
 21. The method of claim 18, wherein said growth factor is anosteoinductive factor.
 22. The method of claim 21, wherein said implantis fitted as a bone filler.
 23. The method of claim 21, wherein saidimplant is fitted in a non-osseous site.
 24. The method of claim 18,wherein said growth factor is a non-osteoinductive factor.